Field of the Technology
The invention relates to the field of laser-scanning based nonlinear optical microscope for rapid imaging of wide areas and large volumes of biological tissues or other materials, ex vivo or in vivo, at sub-micron resolution. Applications include but are not limited to clinical skin imaging, non-invasive skin cancer diagnosis.
Description of the Prior Art
In vivo multiphoton microscopy (MPM) is emerging as an important research and clinical tool for label-free imaging in human skin. The clinical applications of in vivo label-free MPM span from skin cancer detection and diagnosis, to characterizing and understanding keratinocyte metabolism, skin aging, pigment biology, and cosmetic treatments. MPM is based on laser-scanning microscopy, a technique that utilizes a focused laser beam that is raster-scanned across the sample to create high-resolution images. A three dimensional view of the skin can be reconstructed by scanning at multiple depths. Importantly, high-resolution imaging is combined with a label-free contrast mechanism. MPM contrast in skin is derived from second harmonic generation (SHG) of collagen and two-photon excited fluorescence (TPEF) of tissue components such as the co-factors NADH and FAD+, elastin, keratin, and melanin.
Clinical examination crucially relies on the ability to quickly examine large tissue areas and rapidly zoom in to regions of interest. Skin lesions often show irregularity in color and appearance, especially when they start to progress towards malignancy. Imaging of large field of views (FOVs) and automatic translation of the imaging area are critical in the assessment of the entire lesion to avoid false negative diagnosis. Commercial clinical microscopes based on MPM and reflectance confocal microscopy (RCM) have implemented automatic translation of the imaging area. However, the initial FOV is limited to or less than 0.5×0.5 mm2 and thus, assessing large areas of tens of mm2 at different depths may be time consuming and not feasible for clinical use. In an ideal system large FOV and automatic translation of the imaging area would be complemented by fast image acquisition and high detection sensitivity in order for such a system to be of practical utility and efficient use for fast full assessment of skin lesions.
Nonlinear optical (NLO) microscopy comprises a set of imaging techniques that provide high three dimensional resolution and label-free molecular contrast of endogenous components in specimens. NLO microscopy utilizes a focused laser beam that is raster-scanned across the sample to create high-resolution images upon signal detection. A three dimensional-view of the sample can be reconstructed by scanning at multiple depths. Biological tissues are of particular interest owing to NLO microscopy attributes that are tailored for their noninvasive visualization. The list of addressable tissue components includes collagen (through second harmonic generation, SHG), flavin adenosine dinucleotide (FAD), reduced nicotinamide adenine dinucleotide (NADH), keratin, melanin, and elastin fibers (through two-photon excited fluorescence, TPEF), lipids, proteins and water (through coherent Raman scattering, CRS). Endogenous components in biological tissues can also be visualized through third harmonic generation (THG) contrast derived from refractive index discontinuities at interfaces. While this technique does not feature specific molecular contrast, it can be a valuable tool when combined with other imaging modalities, as its higher order nonlinearity and long excitation wavelength provide improved three dimensional-resolution and penetration depth.
The ability to generate high resolution maps of specific tissue molecular compounds without the need for extrinsic labels sets NLO imaging techniques apart from other biomedical imaging methods, and classifies these techniques as preferred tools for label-free imaging of superficial tissues in vivo.
Because of its near-ideal attributes for imaging superficial tissues, NLO microscopy has attracted attention as a high-resolution visualization method of skin in vivo. Koenig et. al. “Flexible Nonlinear Laser Scanning Microscope for Noninvasive Three-Dimensional Detection,” U.S. Pat. No. 9,176,309, discloses a system design for flexible, non-invasive, three-dimensional laser-scanning microscopy using SHG, CARS and multiphoton fluorescence signals such as TPEF from living and non-living matter. A clinical microscope based on this disclosed design has been used in several clinical applications such as skin cancer detection and keratinocyte metabolism assessment. The design includes a scanning unit for two-dimensional deflection of the laser beams, and an image recording based on time-correlated single photon counting (TCSPC).
The disadvantages of the prior design are twofold: 1) The TCSPC detection method is associated with relatively long pixel dwell times (˜20 μs), limiting the scanning speed to a maximum of few seconds per/frame for 512×512 pixels/frame. This detection strategy is not compatible with faster scanning rates that are desirable in many clinical settings; and 2) the close proximity of the mirrors in the scanning unit introduces a motion of the beam at the entrance pupil of the focusing optics that limits the field of view. These shortcomings limit the scanning speed and field of view (FOV). Limited scanning area and slow speed are major limitations for diagnosis and treatment monitoring clinical applications with current technology. Both of those limitations are overcome by the disclosed embodiments of the invention, resulting in a major increase of clinical applicability. Maximizing scanning speed and FOV cannot be achieved in a straightforward manner in the prior art design due to its optical and detection designs as described above.
Advances in the development of NLOM-based microscopes that can image large FOVs have been recently made by several research groups. Prior endeavors have reported on developing an NLOM-based system that can image up to 80 mm2 at a maximum speed of 5 mm/ms by trading-off lateral resolution (between 1.2 μm and 2 μm across the entire FOV). This microscope was applied for imaging resting-state vasomotion across both hemispheres of a murine brain through a transcranial window without the need to stitch adjacent imaging areas. In-depth optimization studies of scan and tube lens designs for minimizing optical aberrations associated with large angle scanning using conventional galvanometer scanning have been produced. Higher scan speeds provided by resonant scanner as the fast axis and conventional galvanometer as the slow axis have been previously implemented in MPM-based systems for several applications, including skin imaging. Neither of prior designs includes all required features for an efficient clinical microscopy imaging device: fast scanning, large FOV, sub-micron resolution.
What is needed therefore for efficient clinical microscopy imaging is an MPM imaging method and apparatus which can image rapidly (<1 μs pixel dwell time), large areas (at least 800×800 μm2) without compromising resolution (sub-micron).